高效液相色谱法测定犬血浆中美洛昔康含量及其在生物等效性研究上的应用

Determination of Meloxicam in Dog Plasma by HPLC and its Application in Bioeqivalence Study

关键词:  美洛昔康  蛋白沉淀法  高效液相色谱法  药动学  生物等效性

Key words:  Meloxicam  Protein Precipitation Method  HPLC  Pharmacokinetics  Bioequivalence

摘要:

建立沉淀蛋白法结合高效液相色谱法灵敏检测犬血浆中美洛昔康含量的分析方法，并将此方法成功应用于美洛昔康在犬体内的药动学和生物等效性研究。血浆样品采用乙腈沉淀蛋白处理后，采用Agilent SB C18（150 mm?4.6 mm，5 μm）色谱柱进行分离，以乙腈-1%甲酸水（80：20，V：V）为流动相进行梯度洗脱，检测波长为355 nm。应用该方法测定18只比格犬随机交叉进行颈部皮下注射0.2 mg/kg·bw的国产制剂和进口制剂的美洛昔康注射剂后的血药浓度，并计算药动学参数。结果显示：美洛昔康在0.050~3.2 μg/mL的浓度范围内呈现良好线性关系（R2?0.999），回收率在98.7%~108.2%以内，批内、批间精密度均小于12%，样品稳定性良好，均满足分析检测要求。单剂量皮下注射美洛昔康注射液受试制剂AUC0-t 、AUC0-∞、Cmax 的90% CI为参比制剂相应参数的80.00%~125.00%范围内，受试制剂和参比制剂具有生物等效性。结果表明，建立的犬血浆中美洛昔康浓度测定方法准确、灵敏、选择性好、重现性高，可用于美洛昔康注射液在犬体内的生物等效性研究。

Abstract:

A promising detection approach, based on protein precipitation method coupled by high performance liquid chromatograph, was established for determination of meloxicam in dog plasma and used in pharmacokinetics and bioequivalence study. Meloxicam was effectively extracted by acetonitrile and separated at the Agilent SB C18 column (150mm?4.6mm, 5μm) using acetonitrile-1% formic acid water (80:20, V:V) as the mobile phase. The UV detector was 355nm. In a randomized crossover study, 18 healthy dogs were administered domestic and imported meloxicam at the dose of 0.2 mg/kg?bw with subcutaneous injection. The results indicated that meloxicam in plasma had a good linear relationship in the range of 0.050~3.2 μg/mL(R2?0.999). The intra-and inter-batch precisions were both less than 12%, the average recoveries was 98.7%~108.2% for spiked plasma，plasma samples were stable during different storage condition. All meet the requirements for analysis and detection.The 90% confidence intervals of AUC0-t,AUC0-∞ and Cmax of the meloxicam injection test preparation after a single dose of subcutaneous injection were within the range of 80.00%~125.00% of the corresponding parameters of the reference preparation.The test preparation and the reference preparation were bioequivalent.The results showed that the established method was accurate, sensitive, selective and reproducible, and suitable for the bioequivalence study of meloxicam injection in dogs.